An investigation of ribonucleoprotein processing in amphibian oocytes

Abstract

Two forms of ribonucleoprotein complex from oocytes of Triturus cristatus carnifex are investigated, heterogeneous nuclear ribonucleoprotein (hnRNP) and a free 40S cytoplasmic RNP particle that stores 5SRNA and transfer RNA in pre-vitellogenic oocytes. HnRNP, isolated from homogenates of Triturus ovaries, has been characterized by sucrose gradient centrifugation, isopycnic centrifugation, electron microscopy and treatment with non-ionic detergent. The results largely confirm previous observations by Malcolm and Sommerville (1974) although electron microscopy of thin-sectioned hnRNP material revealed a considerable degree of cytoplasmic contamination of the preparation. This finding was confirmed by characterization of the polypeptide components of the hnRNP fraction and by comparison with the polypeptide spectrum of manually isolated oocyte nuclei. These studies further revealed that not only were there very few major polypeptides common to both the hnRNP preparation and isolated oocyte nuclei but that the majority of the "hnRNP" polypeptides could be isolated from the oocyte cytoplasm. Comparison of the polypeptide spectra of hnRNP, oocyte nuclei and rat-liver hnRNP "core particles" suggest that a "core protein" homologue may be present in oocyte nuclei though not in the "hnRNP" preparation. Immunostaining of SDS/polyacrylamide gel transfers with an antiserum to rat-liver hnRNP "core protein" revealed the presence of antigenically related polypeptides in the "hnRNP".- It is suggested that a large proportion of the so-called hnRNP preparation from Triturus oocytes could represent partially processed messenger RNP in association with membranous supramolecular structures. The 40S cytoplasmic RNP accumulated in previtellogenic Triturus oocytes contains 5S RNA and transfer RNA with two proteins of molecular mass 45,000 and 39,000 (P45 and P39). The particle has a buoyant density of 1.53 g cm-3 and consists of four identical subunits as shown by salt dissociation and isopycnic centrifugation experiments. Treatment with SDS completely dissociates the RNP complex into its separate components. These can be reassociated into subunits and even intact 40S RNP particles upon removal of the SDS by dialysis. The stable RNA/protein interactions can be demonstrated by analysis of reformed RNP complexes using isopycnic centrifugation and are found to be: 5S RNA/P45, 3(tRNA)/P45, 5S RNA/P39 and 58 RNA/P45/ P39. Indirect immunostaining of frozen oocyte sections with antisera to P45 and P39 suggest a purely cytoplasmic location for P39 whilst P45 is also found in the nucleus. The relationship between the 40S RNP particle proteins with transcription of 5S RNA and transfer RNA is discussed and the possibility that P39 is related to 5S RNA associated ribosomal proteins is also considered. A scheme for the formation and breakdown of 40S RNP storage particles is presented.